Alcoholic liver disease is characterized by steatosis, inflammation, fibrosis and cirrhosis. Interactions among gut-derived bacterial products such as lipopolysaccharide (LPS), metabolites of ethanol, and immune cells contribute to progression of liver injury from steatosis to cirrhosis during chronic alcohol consumption. However, molecular mechanisms underlying these interactions and their associated effects remain poorly understood. It has been shown that normal and efficient tissue repair is critical for liver recovery from injury. Furthermore, efficient removal of dead cells and collagen fibrils is critical for successful tissue repair. This process is governed by several mediators and mediated by macrophages. However, mechanistic insight into the role of these mediators and macrophages in repair of liver injury caused by chronic alcohol consumption is far from complete. Milk fat globule-EGF factor 8 (MFG-E8) is a macrophage-derived glycoprotein. It plays a critical role in promoting tissue repair and diminishing tissue fibrosis in several organs that are composed largely of epithelial cells, but its impact on the liver repair remains unknown. Previously, we and others have demonstrated that severe tissue injury is often associated with down-regulation of MFG-E8 gene expression. Our pilot data showed that (a) MFG-E8 is expressed by macrophages in the liver, (b) metabolites of ethanol and LPS synergistically suppress MFG-E8 gene expression in macrophages, and (c) lack of MFG-E8 causes fibrosis during inflammation in vivo. Based on these preliminary studies, we hypothesized that in addition to causing hepatic steatosis, alcohol and its metabolites synergize with gut-derived bacterial products such as LPS to modulate MFG-E8 gene expression in macrophages, which subsequently influences the outcome of the alcoholic liver disease. This central hypothesis will be tested in three Specific Aims: (1) To examine whether excessive intake of alcohol alters MFG-E8 expression in the liver, if so, to investigate the role of TLR4 (a LPS receptor) and gut-derived LPS in alteration of MFG-E8 gene expression by chronic alcohol consumption. In this study, wild-type and TLR4 mutant mice are fed with ethanol-containing liquid diet, gut-derived LPS is eliminated by treatment with polymyxin B and neomycin, and MFG-E8 gene expression in the liver will be determined with real-time RT-PCR and western blot. (2) To define the molecular mechanism through which metabolites of ethanol and LPS modulate MFG-E8 gene expression in macrophages/Kupffer cells. The standard approach for analysis of the gene expression and promoter function will be applied. The study will be completed through efforts in conjunction with R01DK64240. (3) To study whether alteration of MFG-E8 levels influences the outcome of necroinflammatory liver injury in alcohol-exposed mice injected with LPS. In this study, the cell-specific genetic engineering technology will be used. Together, results from these exploratory data will provide insights into understanding whether and how interactions of alcohol and LPS with MFG-E8 gene in macrophages contribute to progression of alcohol-induced liver damage. They could significantly advance our knowledge about the pathogenesis of alcoholic liver disease. PUBLIC HEALTH RELEVANCE: Alcoholic liver disease affects more than 2 million people in the United States. It is a major public health concern. In this project, we will use novel research tools to investigate molecular mechanisms underlying progression of liver injury induced by chronic alcohol consumption. The results of this exploratory project will have the potential to markedly advance our understanding of underlying pathophysiological causes of progressive alcoholic liver injury. It will ultimately lead to the identification of novel molecular targets and the development of new potential therapeutic agents for patients with alcoholic liver disease.